Anti-sense oligonucleotides are being developed to treat a variety of diseases, particularly viral infections, e.g. Matsukura et al, Proc. Natl. Acad. Sci., Vol. 86, pgs. 4244-4448 (1989). An antisense oligonucleotide is a synthetic oligonucleotide of varying length, usually in the range of about 12 to 30 nucleotides, or nucleotide analogs, whose sequence is complementary to a predetermined segment of RNA, either freshly transcribed or messenger (mRNA), associated with some foreign or otherwise inappropriately expressed gene. It is believed that when an antisense oligonucleotide hybridizes to its target RNA, it either blocks translation or processing of the RNA or makes it susceptible to enzymatic degradation. One problem with this approach has been the difficulty of getting the antisense oligonucleotide to its target RNA in sufficient concentration and for sufficient duration to be effective in shutting down the synthesis of undesired proteins, e.g. viral enzymes, coat proteins, and the like. The susceptibility of the phosphodiester linkage of the oligonucleotides to nuclease digestion is believed to be an important cause of this difficulty, and has prompted the development of a variety of nucleoside oligomers linked by nuclease-resistant analogs of the natural phosphodiester bond, e.g. Miller et al, U.S. Pat. No. 4,511,713 and Ts'o U.S. Pat. No. 4,469,863 (methyl- and arylphosphonates); Miro et al, Nucleic Acids Research, Vol. 17, pgs. 8207-8219 (1989) (phosphoroselenoates); Brill et al, J. Am. Chem. Soc., Vol. 111, pg. 2321 (1989)(phosphorodithioates); and Matsukura et al, Proc. Natl. Acad. Sci., Vol. 84, pgs. 7706-7710 (1987), and Gene, Vol. 72, pgs. 343-347 (1988) (phosphorothioates).
The phosphorothioate and phosphorodithioate analogs are especially promising because they are highly nuclease-resistant, have the same charge as natural oligonucleotides, and are taken up by cells in effective amounts.
Phosphorothioates can be synthesized by automated DNA synthesizers using hydrogen phosphonate or phosphoramidite chemistries. In the former approach, the phosphonate backbone can be sulfurized in a single step off of the automated synthesizer after synthesis. This is advantageous because the phosphonate moieties are sulfurized by exposure to elemental sulfur dissolved in an organic solvent. Since the sulfur readily precipitates out of solution, the off-column sulfurization avoids costly blockages of valves and tubing of the synthesizer by sulfur precipitates. A drawback of this route of phosphorothioate synthesis is that coupling yields during chain elongation are typically lower than those obtained using phosphoramidite chemistry, Gaffney and Jones, Tett. Lett., Vol. 29, pgs. 2619-2622 (1988). The practical importance of high coupling yields is demonstrated by the synthesis of a 28-mer where a 99% coupling yield per step results in an overall yield of 76% (0.99.sup.27), whereas a 96% yield per step results in an overall yield of only 33% (0.96.sup.27).
Phosphoramidite chemistry, with coupling yields typically greater than 99%, is presently the more desirable approach to phosphorothioate and phosphorodithioate synthesis. However, the phosphite intermediates, which would be sulfurized, are unstable under the conditions of the detritylization step of the reaction cycle. Thus, the phosphite linkage must be sulfurized after each coupling step. This can be accomplished with a variety of sulfurizing agents, e.g. Matsukura et al, Gene (cited above)(elemental sulfur); lyer et al, J. Org. Chem., Vol. 55, pgs. 4693-4699 (1990)(a thiosulfonate sulfurizing agent); Hirschbein, U.S. patent application Ser. No. 07/464,182 (thiuram disulfide and polysulfide sulfurizing agents); and Stec et al, U.S. patent application Ser. No. 07/512,644 (thiophosphorus sulfurizing agents). Unfortunately, none of these agents provides 100% sulfurization. At each sulfurization step a small fraction of the phosphite precursors are oxygenated instead of sulfurized. This leads to the synthesis of a complex mixture of phosphorothioate oligonucleotides with respect to the number and distribution of oxygens along the phosphodiester backbone. The fraction of a product containing a given number of oxygens follows the binomial distribution. For example, in the synthesis of a 20-mer phosphorothioate oligonucleotide where the sulfurization yield is 99% at each step, the fraction of the product with, say, 1 and 2 oxygenations in place of sulfurizations is given by the second and third terms, respectively, of the binomial expansion of (0.99+0.01).sup.20, or (.sub.1.sup.20) (0.99).sup.19 (0.01)=0.165 and (.sub.2.sup.20)(0.99).sup.18 (0.01).sup.2 =0.016, respectively. Thus, relatively large fractions of even modestly sized phosphorothioate oligonucleotides are incompletely sulfurized and, because of physiochemical similarity of the completely and incompletely sulfurized compounds, separation and/or analysis of the two species has proven to be inconvenient, usually requiring NMR analysis, or like procedures.
In view of the desire to employ phosphorothioate and phosphorodithioate analogs of oligonucleotides as pharmaceutical compounds, it would be advantageous to have available methods for preparation and analysis of the sulfurized products that would permit separation of fully sulfurized species from partially sulfurized species and that would permit a convenient and inexpensive way of monitoring yields of completely sulfurized analogs, particularly in connection with phosphoramidite and/or phosphorthioamidite chemistries.